Nitisinone for treatment of oculocutaneous/ocular albinism and for increasing pigmentation

ABSTRACT

A method is provided for the treatment of vision problems in a subject suffering from one of various forms of albinism, including, for example, oculocutaneous albinism types OCA1a and OCA1b, as well as ocular albinism type 1, resulting from mutations in the GPR143 gene, as well as the OCA2, OCA3 or OCA4 genes, by administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the compound (2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione), also known as NTBC for a sufficient period of time. The administration of NTBC is believed to increase the amount of pigmentation in the subject and alleviate certain symptoms caused by lack of pigmentation in the eye tissues. Also described are methods of use of NTBC for increasing the pigmentation of a subject for cosmetic purposes, by administering to the subject a therapeutically effective amount of NTBC.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/308,771, filed on Feb. 26, 2010, the entire contents of which areincorporated by reference.

BACKGROUND OF THE INVENTION

Albinism (also called achromia, achromasia, or achromatosis) is acongenital disorder characterized by the complete or partial absence ofpigment in the skin, hair, and eyes due to absence or defect of anenzyme involved in the production of melanin. Certain forms of albinismare known to be due to mutations in tyrosine metabolism. Albinismresults from inheritance of recessive gene alleles and is known toaffect all vertebrates, including humans. There is also known anX-linked form of albinism. Patients with albinism have significantvisual disability.

In oculocutaneous albinism (OCA) (despite its Latin-derived name meaning“eye-and-skin” albinism), pigment is lacking in the eyes, skin, andhair. (The equivalent mutation in non-humans also results in lack ofmelanin in the fur, scales, or feathers.) People with oculocutaneousalbinism can have anything from no pigment at all to near normal levelsof pigmentation. There are at least three general types of OCA,characterized as Type 1, Type 2 and Type 3.

Oculocutaneous albinism type 1 (OCA1) is caused by a mutation in thetyrosinase gene, and can occur in two variations. Tyrosinase convertstyrosine to dihydroxyphenylalanine (DOPA) and DOPAquinone. The firsttype OCA1 mutation found was identified as OCA1a, resulting in anorganism that cannot develop pigment at all. The hair is usually white(often translucent) and the skin very pale. Vision in an affectedindividual usually ranges from 20/200 to 20/400. A second known typeOCA1 mutation is identified as type OCA1b, which itself has severalsubtypes. This is a less severe form of albinism and some affectedindividuals with OCA1b can actually tan and develop pigment in the hair.

Patients with albinism experience varying degrees of vision lossassociated with foveal hypoplasia, nystagmus, photophobia and/or glaresensitivity, refractive errors, and abnormal decussation of ganglioncell axons at the optic chiasm. Current treatment options for visionproblems caused by albinism are limited to correction of refractiveerrors and amblyopia, low vision aids, and (in some cases) extraocularmuscle surgery.

Another form of albinism is ocular albinism. Ocular albinism is agenetic condition that primarily affects the eyes. In ocular albinism,only the eyes lack pigment. People who have ocular albinism havegenerally normal skin and hair color, although their coloration istypically lighter than either parent. Many even have a normal eyeappearance. This condition reduces the coloring (pigmentation) of theiris, which is the colored part of the eye, and the retina, which is thelight-sensitive tissue at the back of the eye. Pigmentation in the eyeis essential for normal vision.

Ocular albinism is characterized by severely impaired sharpness ofvision (visual acuity) and problems with combining vision from both eyesto perceive depth (stereoscopic vision). Although the vision loss ispermanent, it does not worsen over time. Other eye abnormalitiesassociated with this condition include rapid, involuntary eye movements(nystagmus), eyes that do not look in the same direction (strabismus),and increased sensitivity to light (photophobia). Many affectedindividuals also have abnormalities involving the optic nerves, whichcarry visual information from the eye to the brain.

Unlike some other forms of albinism, ocular albinism does notsignificantly affect the color of the skin and hair. People with thiscondition may have a somewhat lighter complexion than other members oftheir family, but these differences are usually minor.

Ocular albinism type 1 results from mutations in the GPR143 gene. Thisgene is responsible for making a protein that plays a role inpigmentation of the eyes and skin. The GPR143 gene helps control thegrowth of melanosomes, which are cellular structures that produce andstore a pigment called melanin. Melanin is the substance that givesskin, hair, and eyes their color. In the retina, this pigment also playsa role in normal vision.

Most mutations in the GPR143 gene alter the size or shape of the GPR143protein. Many of these genetic changes prevent the protein from reachingmelanosomes to control their growth. In other cases, the protein reachesmelanosomes normally, but mutations disrupt the protein's function. As aresult of these changes, melanosomes in skin cells and the retina cangrow abnormally large. Researchers are uncertain how these giantmelanosomes are related to vision loss and other eye abnormalities inpeople with ocular albinism.

Currently, treatments for the visual impairment of oculocutaneousalbinism are quite limited, and children with OCA may be left withvision approaching or reaching legal blindness. Even a modest effect onvisual function (such as reduction of glare and light sensitivity) isgreatly appreciated by patients.

As such, there exists a need for improving the treatment of patients,particularly for improving the vision of patients, suffering fromvarious forms of albinism.

BRIEF SUMMARY OF THE INVENTION

Nitisinone (NTBC) is an FDA-approved drug used in the treatment oftyrosinemia, type 1. The drug blocks the normal degradation pathway oftyrosine, thus allowing greater circulating plasma levels of tyrosine.In accordance with the present invention, it was found thatadministration of NTBC to subjects (e.g., mice or humans) with certainforms of albinism, resulted in increased circulating tyrosine levels, anincrease in tyrosinase activity, and, subsequently, increasedpigmentation.

In an embodiment, the present invention provides a method for increasingtyrosine plasma concentrations in a subject suffering fromoculocutaneous albinism, the method comprising administering to thesubject a pharmaceutically acceptable composition comprising NTBC in atherapeutically effective amount. In an embodiment, an effective amountof NTBC is the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from about 7micromolar (μM) to about 2 millimolar (mM). In a further embodiment, aneffective amount of NTBC is the amount administered to a subject thatresults in plasma concentrations of tyrosine in the subject increasingfrom about 70 μM to about 2 mM. In another embodiment, an effectiveamount of NTBC is the amount administered to a subject that results inplasma concentrations of tyrosine in the subject increasing from atleast about 50 μM to a range of about 300 μM. In a preferred embodiment,an effective amount of NTBC is the amount administered to a subject thatresults in plasma concentrations of tyrosine in the subject increasingfrom at least about 70 μM. In another embodiment, the therapeuticallyeffective amount of NTBC administered to a subject is at least about 0.1mg/kg/day, in some embodiments, in the range of between about 0.1mg/kg/day to about 10 mg/kg/day. Preferably, in another embodiment, thetherapeutically effective amount of NTBC administered to a subject is atleast about 0.5 mg/kg/day to about 4 mg/kg/day. In another embodiment,the therapeutically effective amount of NTBC administered to a subjectis about 1 mg/kg/day to about 2 mg/kg/day, preferably about 1 mg/kg/day.

In another embodiment, the present invention provides a method forincreasing tyrosine plasma concentrations in a subject suffering fromoculocutaneous albinism, wherein the albinism is identified as typeOCA1a, or type OCA1b.

In another embodiment, the present invention provides a method forincreasing tyrosine plasma concentrations in a subject suffering fromocular albinism, the method comprising administering to the subject apharmaceutically acceptable composition comprising NTBC in atherapeutically effective amount. In an embodiment, an effective amountof NTBC is the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from about 7 μM toabout 2 mM. In a further embodiment, an effective amount of NTBC is theamount administered to a subject that results in plasma concentrationsof tyrosine in the subject increasing from about 70 μM to about 2 mM. Inanother embodiment, an effective amount of NTBC is the amountadministered to a subject that results in plasma concentrations oftyrosine in the subject increasing from at least about 50 μM to a rangeof about 300 μM. In a preferred embodiment, an effective amount of NTBCis the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from at least about70 μM. In another embodiment, the therapeutically effective amount ofNTBC administered to a subject is at least about 0.1 mg/kg/day, in someembodiments, in the range of between about 0.1 mg/kg/day to about 10mg/kg/day. Preferably, in another embodiment, the therapeuticallyeffective amount of NTBC administered to a subject is at least about 0.5mg/kg/day to about 4 mg/kg/day. In another embodiment, thetherapeutically effective amount of NTBC administered to a subject isabout 1 mg/kg/day to about 2 mg/kg/day, preferably about 1 mg/kg/day.

In yet another embodiment, the present invention provides a method fortreating impaired vision in a subject suffering from oculocutaneousalbinism, or ocular albinism, the method comprising administering to thesubject a pharmaceutically acceptable composition comprising NTBC in atherapeutically effective amount. In an embodiment, an effective amountof NTBC is the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from about 7 μM toabout 2 mM. In a further embodiment, an effective amount of NTBC is theamount administered to a subject that results in plasma concentrationsof tyrosine in the subject increasing from about 70 μM to about 2 mM. Inanother embodiment, an effective amount of NTBC is the amountadministered to a subject that results in plasma concentrations oftyrosine in the subject increasing from at least about 50 μM to a rangeof about 300 μM. In a preferred embodiment, an effective amount of NTBCis the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from at least about70 μM. In another embodiment, the therapeutically effective amount ofNTBC administered to a subject is at least about 0.1 mg/kg/day, in someembodiments, in the range of between about 0.1 mg/kg/day to about 10mg/kg/day. Preferably, in another embodiment, the therapeuticallyeffective amount of NTBC administered to a subject is at least about 0.5mg/kg/day to about 4 mg/kg/day. In another embodiment, thetherapeutically effective amount of NTBC administered to a subject isabout 1 mg/kg/day to about 2 mg/kg/day, preferably about 1 mg/kg/day.

In a further embodiment, the present invention provides a method forincreasing pigmentation in the eyes, hair and/or skin of a subject, themethod comprising administering to the subject a pharmaceuticallyacceptable composition comprising NTBC in a therapeutically effectiveamount such that the plasma concentrations of tyrosine in the subjectare increased to an amount sufficient to increase visually discernablepigmentation in the subject.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 is the chemical structure of NTBC.

FIG. 2 shows the metabolic pathway for tyrosine degradation in mammalsand where NBTC blocks the enzyme 4-hydroxyphenyl-pyruvate dioxygenaseearly in the pathway.

FIG. 3 shows a comparison of electron micrographs of retinal pigmentepithelial (RPE) cells from C57BL/6J-Tyr^(c-h/c-h) mice, which are amodel for oculocutaneous albinism. The micrographs show increases inmelanosomes (dark circles) in the mice treated with NTBC when comparedto controls.

FIG. 4 is a comparison of the number of pigmented melanosomes in oculartissues of vehicle- and NTBC-treated C57BL/6-Tyr^(c-h/c-h) (Himalayan)mice.

FIG. 5 is a graph showing the steady state levels of wild-typetyrosinase, stabilized relative to GAPDH at various time points by 1 mMtyrosine.

FIG. 6 is a graph showing the steady state levels of R77L tyrosinase,stabilized relative to GAPDH at various time points by 1 mM tyrosine.

FIG. 7 is a graph showing the steady state levels of H420R tyrosinase,stabilized relative to GAPDH at various time points by 1 mM tyrosine.The stability of the H420R—but not the R77L—protein is stabilizedrelative to GAPDH at later time points (9 and 12 hours) by 1 mMtyrosine. Two-tailed test of significance: p-value <0.0001 (**), p-value<0.05 (*).

FIG. 8 shows that increased ambient tyrosine promotes pigment productionin OCA-1B, but no OCA-1A, allele-expressing cells. (8A) Although Melan-ccells transfected with WT, R77L, or H420R tyrosinase express comparablelevels of protein (inset), only the H420R tyrosinase responds to 1 mMtyrosine by increasing pigment production. (8B, 8C) Cultured humanmelanocytes from control and OCA1-B, but not OCA-1A patients showincreased pigment on incubation with 1 mM tyrosine.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the present invention provides a method for increasingtyrosine plasma concentrations in a subject suffering fromoculocutaneous albinism, the method comprising administering to thesubject a pharmaceutically acceptable composition comprising NTBC in atherapeutically effective amount. In an embodiment, an effective amountof NTBC is the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from about 7 μM toabout 2 mM. In a further embodiment, an effective amount of NTBC is theamount administered to a subject that results in plasma concentrationsof tyrosine in the subject increasing from about 70 μM to about 2 mM. Inanother embodiment, an effective amount of NTBC is the amountadministered to a subject that results in plasma concentrations oftyrosine in the subject increasing from at least about 50 μM to a rangeof about 300 μM. In a preferred embodiment, an effective amount of NTBCis the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from at least about70 μM. In another embodiment, the therapeutically effective amount ofNTBC administered to a subject is at least about 0.1 mg/kg/day, in someembodiments, in the range of between about 0.1 mg/kg/day to about 10mg/kg/day. Preferably, in another embodiment, the therapeuticallyeffective amount of NTBC administered to a subject is at least about 0.5mg/kg/day to about 4 mg/kg/day. In another embodiment, thetherapeutically effective amount of NTBC administered to a subject isabout 1 mg/kg/day to about 2 mg/kg/day, preferably about 1 mg/kg/day.

In another embodiment, the present invention provides a method forincreasing tyrosine plasma concentrations in a subject suffering fromoculocutaneous albinism, for example, wherein the albinism is identifiedas type OCA1a, meaning the affected subject has no measurable tyrosinaseactivity, or type OCA1b, meaning the affected subject has greatlydiminished tyrosinase activity. It is contemplated that other forms ofoculocutaneous albinism can be treated by the methods of the presentinvention.

In yet another embodiment, the present invention provides a method forincreasing tyrosine plasma concentrations in a subject suffering fromocular albinism, the method comprising administering to the subject apharmaceutically acceptable composition comprising NTBC in atherapeutically effective amount. In an embodiment, an effective amountof NTBC is the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from about 7 μM toabout 2 mM. In a further embodiment, an effective amount of NTBC is theamount administered to a subject that results in plasma concentrationsof tyrosine in the subject increasing from about 70 μM to about 2 mM. Inanother embodiment, an effective amount of NTBC is the amountadministered to a subject that results in plasma concentrations oftyrosine in the subject increasing from at least about 50 μM to a rangeof about 300 μM. In a preferred embodiment, an effective amount of NTBCis the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from at least about70 μM. In another embodiment, the therapeutically effective amount ofNTBC administered to a subject is at least about 0.1 mg/kg/day, in someembodiments, in the range of between about 0.1 mg/kg/day to about 10mg/kg/day. Preferably, in another embodiment, the therapeuticallyeffective amount of NTBC administered to a subject is at least about 0.5mg/kg/day to about 4 mg/kg/day. In another embodiment, thetherapeutically effective amount of NTBC administered to a subject isabout 1 mg/kg/day to about 2 mg/kg/day, preferably about 1 mg/kg/day.

In yet another embodiment, the present invention provides a method fortreating impaired vision in a subject suffering from oculocutaneousalbinism, or ocular albinism, the method comprising administering to thesubject a pharmaceutically acceptable composition comprising NTBC in atherapeutically effective amount. In an embodiment, an effective amountof NTBC is the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from about 7 μM toabout 2 mM. In a further embodiment, an effective amount of NTBC is theamount administered to a subject that results in plasma concentrationsof tyrosine in the subject increasing from about 70 μM to about 2 mM. Inanother embodiment, an effective amount of NTBC is the amountadministered to a subject that results in plasma concentrations oftyrosine in the subject increasing from at least about 50 μM to a rangeof about 300 μM. In a preferred embodiment, an effective amount of NTBCis the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from at least about70 μM. In another embodiment, the therapeutically effective amount ofNTBC administered to a subject is at least about 0.1 mg/kg/day, in someembodiments, in the range of between about 0.1 mg/kg/day to about 10mg/kg/day. Preferably, in another embodiment, the therapeuticallyeffective amount of NTBC administered to a subject is at least about 0.5mg/kg/day to about 4 mg/kg/day. In another embodiment, thetherapeutically effective amount of NTBC administered to a subject isabout 1 mg/kg/day to about 2 mg/kg/day, preferably about 1 mg/kg/day.

In a further embodiment, the present invention provides a method oftreating oculocutaneous albinism, or ocular albinism, the methodcomprising administering to the subject a pharmaceutically acceptablecomposition comprising NTBC in a therapeutically effective amount. Inaddition, the methods disclosed herein are not limited to oculocutaneousalbinism, or ocular albinism, but can be used for subjects having otherforms of albinism, including, but not limited to, for example, albinismwhich results from mutations in the microtubulin associated protein tau(MATP, OCA4) gene, the P protein gene (OCA2) (See, Brilliant, M. H.,Pigment Cell Res., 14:86-93 (2001)), and in the tyrosine related protein1 (TYRP1, OCA3) gene (See, Sarangarajan, R. et al., Pigment Cell Res.,14:437-44 (2001)).

In a further embodiment, the present invention provides a method forincreasing pigmentation in the eyes, hair, and/or skin of a subject, themethod comprising administering to the subject a pharmaceuticallyacceptable composition comprising NTBC in a therapeutically effectiveamount such that the plasma concentrations of tyrosine in the subjectare increased to an amount sufficient to increase visually discernablepigmentation in the subject. In this embodiment, the use of NTBC toincrease skin, eye, or hair pigmentation, is for cosmetic purposes.

In accordance with the present invention, in an embodiment, the presentinvention provides a pharmaceutical composition comprising 2-(−2-nitro-4trifluoromethylbenzoyl)-1,3 cyclohexanedione (NTBC) or apharmaceutically acceptable salt, hydrate, or solvate thereof, whereinthe composition includes a pharmaceutically and physiologicallyacceptable carrier, in an amount effective for use in a medicament,preferably for use as a medicament for treating impaired vision in theeyes of a subject suffering from oculocutaneous albinism, or for use asa medicament for treating impaired vision in the eyes a subjectsuffering from ocular albinism, or for use as a medicament forincreasing pigmentation in the eyes, hair, or skin of a subject, whenadministered to the subject in an effective amount. In an embodiment, aneffective amount of NTBC is the amount administered to a subject thatresults in plasma concentrations of tyrosine in the subject increasingfrom about 7 μM to about 2 mM. In a further embodiment, an effectiveamount of NTBC is the amount administered to a subject that results inplasma concentrations of tyrosine in the subject increasing from about70 μM to about 2 mM. In another embodiment, an effective amount of NTBCis the amount administered to a subject that results in plasmaconcentrations of tyrosine in the subject increasing from at least about50 μM to a range of about 300 μM. In a preferred embodiment, aneffective amount of NTBC is the amount administered to a subject thatresults in plasma concentrations of tyrosine in the subject increasingfrom at least about 70 μM. In another embodiment, the therapeuticallyeffective amount of NTBC administered to a subject is at least about 0.1mg/kg/day, in some embodiments, in the range of between about 0.1mg/kg/day to about 10 mg/kg/day. Preferably, in another embodiment, thetherapeutically effective amount of NTBC administered to a subject is atleast about 0.5 mg/kg/day to about 4 mg/kg/day. In another embodiment,the therapeutically effective amount of NTBC administered to a subjectis about 1 mg/kg/day to about 2 mg/kg/day, preferably about 1 mg/kg/day.

It is also contemplated that the pharmaceutical composition of thepresent invention can be used to treat subjects having type OCA1aoculocutaneous albinism, and/or type OCA1b oculocutaneous albinismand/or type 1 ocular albinism.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising 2-(−2-nitro-4 trifluoromethylbenzoyl)-1,3cyclohexanedione (NTBC) or a pharmaceutically acceptable salt, hydrate,or solvate thereof, wherein the composition includes a pharmaceuticallyand physiologically acceptable carrier, in an amount effective for usein a medicament, preferably for use as a medicament for treatingimpaired vision in the eyes of a subject suffering from oculocutaneousalbinism, or for use as a medicament for treating impaired vision in theeyes a subject suffering from ocular albinism, or for use as amedicament for increasing pigmentation in the eyes, hair, or skin of asubject, when administered to the subject in an effective amount, whenthe subject is suffering from albinism due to a mutation in the Pprotein gene (OCA2), and/or the tyrosinase-related protein-1 (TYRP-1,OCA3), and/or the microtubulin associated protein tau (MATP, OCA4) gene.

NTBC is marketed as Orfardin®, which was designated an orphan drug inMay 1995 by the FDA, for the treatment of a rare inherited disorder ofintermediate metabolism, type 1 tyrosinemia, in which patients areunable to properly break down the amino acid tyrosine (FIG. 1). Thesynthesis and use of NTBC, and its related compounds, as an herbicide,is described in U.S. Pat. No. 5,006,158.

NTBC blocks the enzyme parahydroxyphenylpyruvic acid dioxygenase(p-HPPD), the second step in the tyrosine degradation pathway, preventsthe accumulation of fumarylacetoacetate and its conversion tosuccinylacetone (FIG. 2).

Because NTBC increases the concentration of tyrosine in the blood,dietary management with controlled intake of phenylalanine and tyrosineshould considered immediately upon diagnosis, to prevent tyrosinecrystals from forming in the cornea of subjects undergoing NTBC therapy.If the blood concentration of phenylalanine becomes too low (<20 μM),additional protein should be added to the diet.

In those embodiments where the route of administration is other thanoral, the therapeutic compositions of the present invention generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle. The route of administration of NTBC, inaccordance with the present invention, is in accord with known methods,e.g., oral ingestion, injection or infusion by intravenous,intraperitoneal, intramuscular, intrarterial, subcutaneous,intralesional routes, by aerosol or intranasal routes, or by sustainedrelease systems as noted below. NTBC can be administered continuously byinfusion or by bolus injection.

The term “treat” as well as words stemming therefrom, as used herein, donot necessarily imply 100% or complete treatment. Rather, there arevarying degrees of treatment of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect. In thisrespect, the inventive methods can provide any amount of any level oftreatment of albinism in a subject. Furthermore, the treatment providedby the inventive method can include treatment of one or more conditionsor symptoms of the albinism being treated.

An effective amount of NTBC to be employed therapeutically will depend,for example, upon the therapeutic and treatment objectives, the route ofadministration, the age, condition, and body mass of the patientundergoing treatment or therapy, and auxiliary or adjuvant therapiesbeing provided to the patient. Accordingly, it will be necessary androutine for the practitioner to titer the dosage and modify the route ofadministration, as required, to obtain the optimal therapeutic effect. Atypical daily dosage might range from at least about 0.1 mg/kg/day to upto about 100 mg/kg/day or more, preferably from about 0.1 to about 10mg/kg/day depending on the above-mentioned factors. Typically, theclinician will administer antibody until a dosage is reached thatachieves the desired effect. The progress of this therapy is easilymonitored by conventional assays.

The dosage ranges for the administration NTBC are those large enough toproduce the desired effect in which the visual symptoms of albinism,such as nystagmus, photosensitivity or strabismus are ameliorated. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof disease of the patient and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any complication. In an embodiment, the methods of the presentinvention provide for the administration of NTBC to children sufferingfrom albinism, ranging in age from six months to 6 years old. In anotherembodiment, the methods of the present invention provide for theadministration of NTBC to adults suffering from albinism.

NTBC is generally prescribed to patients suffering from oculocutaneousalbinism or ocular albinism at dosages of about 1.0 mg/kg/day; however,individual doses may vary. For example, dosages may be from at leastabout 0.1 mg/kg/day to about 10 mg/kg/day, or preferably, from about 0.5mg/kg/day to about 5 mg/kg/day. Dosage should be adjusted to maintainplasma tyrosine concentrations between at least about 10 μM, toconcentrations in the millimolar range. For example, plasma tyrosineconcentrations between at least about 50 μM, to about 2 mM, or forexample, plasma tyrosine concentrations between at least about 70 toabout 1 mM, or for example, plasma tyrosine concentrations between about200 μM to about 500 μM. It is contemplated that the therapeuticallyeffective dosage is one that theoretically blocks greater than 99%ofp-HPPD activity.

NTBC can be administered orally, intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally, alone orin combination with other drugs. Preferably, NTBC is administered orallyby capsule or pill form. It is understood that the pills formulated fororal administration, including pills used in the present invention, maycontain ingredients to serve as fillers, binders and for color codingpurposes. These ingredients are in common use in many oral formulationsand may include, but are not limited to, lactose, corn starch, calciumphosphate, povidone, magnesium stearate, stearic acid, colloidal silicondioxide, hydroxypropyl methylcellulose, polyethylene glycol and one ormore of the following dyes: FD&C Blue No. 1 Lake, FD&C Blue No. 2Aluminum Lake, D&C Green No. 5, D&C Yellow No. 10, FD&C Yellow No. 6 orFD&C Red No. 3. Of course, these are only exemplary fillers and dyes,those of skill in the art will recognize that other inactive ingredientsmay be used in the preparation of the formulations of the presentinvention.

Preparations for parenteral administration include, for example, sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include, for example, water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include, for example, sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include, forexample, fluid and nutrient replenishers, electrolyte replenishers (suchas those based on Ringer's dextrose), and the like. Preservatives andother additives may also be present such as, for example,antimicrobials, anti-oxidants, chelating agents, and inert gases and thelike.

Injectable formulations are also in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630(2009)).

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with theactive compound(s), and by the route of administration. Thepharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents, are well-known to thoseskilled in the art and are readily available to the public. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active agent(s) and one which has no detrimentalside effects or toxicity under the conditions of use.

Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain, forexample, one or more nonionic surfactants having a hydrophile-lipophilebalance (HLB) of illustratively, from about 12 to about 17. The quantityof surfactant in such formulations will typically range from about 5% toabout 15% by weight. Suitable surfactants include, for example,polyethylene glycol sorbitan fatty acid esters, such as sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampoules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid excipient, for example, water, forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules, andtablets.

In an embodiment, NTBC is given orally in two divided doses; however,because of the long half-life (50-60 hours), affected individuals whoare older and more stable may maintain adequate therapy withonce-per-day dosing. For example, dosages can be given once a day, oronce every other day.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Animal Husbandry and Clinical Examination. C57BL/6J mice (Stock#000664), C57BL/6JTyr^(c-2J/c-2J) mice (Stock #000058, MGI ID#1855985),and C57BL/6Tyr^(c-h/c-h) (Stock #000104, MGI ID#1855979) were obtainedfrom the Jackson Laboratory (Bar Harbor, Me.). Mice were housedaccording to the institutional Animal Review Board standards with a 14hour light/10 hour dark cycle. These studies conformed to the principlesfor laboratory animal research outlined by the Animal Welfare Act(NIH/HHS) and the ARVO Statement for the Use of Animals in Ophthalmicand Vision Research and were approved by the Institutional Animal Careand Use Committee of the National Eye Institute. Clinical examinationand imaging of the anterior segment of mice were performed ongently-restrained, awake mice using a Haag-Streit BQ slit lamp andImaging Module IM900® software (Haag-Streit, Inc., Mason, Ohio).Clinical examination of the posterior segment was performed on gentlyrestrained, awake mice after dilation with one drop of 1% tropicamide(Alcon Laboratories, Inc., Fort Worth, Tex.) using an indirectophthalmoscope (Keeler, Windsor, Berkshire, UK) with a 90D condensinglens (Volk, Mentor, Ohio). Fundus images were obtained on mice sedatedwith intraperitoneally injected 100 mg/ml ketamine, 200 mg/ml xylazinediluted in normal saline. Images were obtained using a Nikon D.90digital SLR camera with a Nikon 85 mm f/2.8D micro AF-S ED lens mountedto a custom-made aluminum stand, using a 5 cm long Hopkins rigidotoscope coupled to a Xenon Nova light source (175 watt) and fiberopticcable (Karl Storz, Tuttlingen, Germany). Mice were euthanized withcarbon dioxide according to institutional guidelines.

Drug Dosing and Monitoring. Ten C57BL/6J Tyr^(c-2J/c-2J) and tenC57BL/6Tyr^(c-h/c-h) mice, 3-4 months of age were designated fortreatment with NTBC (Swedish Orphan Drug, Stockholm, Sweden). An equalnumber of age-matched controls of each genotype designated to receivevehicle treatment. NTBC was dissolved in 2 M NaOH and brought to neutralpH before administration to mice. Coat color, iris transillumination andfundus appearance were photodocumented prior to treatment. Becausepigment deposition in hair is stimulated with new hair growth, a sectionof each mouse's coat was shaved prior to the beginning of theexperiment. Drug or vehicle was given every other day via oral gavages,at a dosage of about 4 mg/kg, in a volume of about 0.2-0.3 ml. This doseof NTBC was chosen to give plasma tyrosine concentrations in the rangeof about 0.3-0.7 mM, or approximately two to four times the dosestypically used in humans with tyrosinemia type 1, and within the limitsof the maximally tolerated dose in mice. Coat color, iristransillumination and fundus appearance were photodocumented at the endof 1 month of treatment or vehicle dosing. For prenatal treatmentexperiments, pregnancy was determined by a maternal weight gain of >2 gover 7-9 days, after observing a vaginal mucus plug. Treatment with 4mg/kg NTBC was initiated daily at day 9 or 10 of pregnancy by oralgavage, and given until birth of the litter. At that point,every-other-day oral treatment of the mother was initiated until time ofweaning.

Plasma tyrosine was assayed from retro-orbital blood taken from mice at1 week, and 4 weeks into treatment. Because the volume of blood thatcould be obtained from non-terminal bleeds was small, the plasma from2-3 mice was pooled to make a single measurement. Plasma samples werefrozen immediately after collection on dry ice. Samples ready for assaywere gently thawed, diluted with an equal volume of loading buffer (0.2M lithium citrate, pH 2.2), and filtered using Vivaspin 500 (3000 Damolecular weight cut-off, Sartorius Stedman Biotech, Goettingen,Germany) spun in a fixed angle centrifuge at about 14000×g for 60minutes at a temperature of about 16-20° C. The supernatant wascollected and tyrosine was quantitated on a Biochrom 30spectrophotometer (Biochrom, Ltd., Cambridge, UK) using themanufacturer's specifications.

Transmission Electron Microscopy. Eyes from drug and vehicle-treatedmice were dissected and divided into anterior and posterior segments(n=2 eyes from 2 separate mice in each group). The iris and posteriorpart (choroid, RPE and retina) of the eyes were removed and fixed in 2%glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer(PB), pH 7.4 for about 12 hours at room temperature (RT). After a washwith rinsing buffer (RB, 4% sucrose and 0.15 mM CaCl₂ in PB), pH 7.4 at4° C., tissues were postfixed in 1% OsO₄ in 0.1 M PB, pH 7.4 for 1 hour.After rinsing and dehydration, tissues were embedded in Durcupan resinfor 72 hours at 60° C. One micron semi-sections were used for tissueorientation. About 70-90 nm ultrasections were also collected in 200mesh grids and counterstained with 5% uranyl acetate and 0.3% leadcitrate. Sections were viewed on a JEOL 1010 transmission electronmicroscope at 60 KV (JEOL Korea, Ltd., Seoul, KR) and digital imageswere acquired at about 8000× to about 30,000× magnification by AMTsoftware (Advanced Microscopy Techniques, Corp., Danvers, Mass.).

Structural Modeling. The atomic structures of the mouse tyrosinase havebeen modeled using the crystal coordinates of two bicopper-bindingtyrosinase proteins from the RCSB protein data bank(http://www.pdb.org/pdb) as structural templates: 1) StreptomycesCastaneoglobisporus tyrosinase complexed with a caddie protein [ProteinData Bank ID: 2ah1]; and 2) the Ipomea Batatas sweet potato catechol(O-diphenol) oxidase containing dicopper center [Protein Data Bank ID:1bt3]) (E. Abola, et al., in Crystallographic Ddatabases-InformationContent, Software Systems, Scientific Applications, G. Bergerhoff, R.Sievers, Eds. (Data Comission on the International Union ofCrystallography, Cambridge, 1987), pp. 107-132). Briefly, the structuralalignment of these proteins was performed using the MatchMaker moduleincorporated in the UCSF Chimera, build 1.4.1 (E. F. Pettersen et al.,J. Comput. Chem., 25:1605 (2004)). Primary sequences were aligned usingthe method of Needleman and Wunsch (S. B. Needleman & C. D. Wunsch, J.Mol. Biol., 48:443 (1970)) integrated in the program Look, version 3.5.2for tertiary structure prediction (C. Lee, J. Mol. Biol., 236:918(1994)). The conformation of the missense variants, R77L and H420R, wasgenerated by the same program implicating a self-consistent ensembleoptimization (500 cycles).

In Vitro Expression and Enzyme Activity. The expression construct ofmouse Tyr was a kind gift of Dr. C. Olivares from the School of Medicineof the University of Murcia (Spain). This construct was prepared in thepcDNA3.1 expression vector (Invitrogen, Carlsbad, Calif.) usingEcoRI/XbaI restriction sites and based on the mouse Tyr clone obtainedas described previously in C. Olivares, et al., Biochem. J., 354:131(2001). Constructs of tyrosinase gene mutant variants, with changescorresponding to missense mutations R77L and H420R in the mousetyrosinase protein sequence, were created using standard methodologies(Mutagenex Inc., Piscataway, N.J.). All mutational changes were verifiedby the cDNA sequencing. Protein lysates for the wild type mousetyrosinase, R77L and H420R missense variants were electrophoreticallyseparated under reducing conditions, blotted, and then probed with αPEP7antiserum, directed against the C-terminal cytosolic extension of Tyr.The αPEP7 antiserum was a generous gift from Dr. V. J. Hearing from theNational Cancer Institute, National Institutes of Health, Bethesda, Md.

Chinese hamster ovary cells (CHO) (generous gift from Dr. J. T.Wroblewski from the Pharmacology Department, Georgetown UniversityMedical Center, Washington D.C.) were grown at a temperature of about37° C. in DMEM media, in the presence of 10% fetal bovine serum, 1%penicillin and 4.5 g proline per 0.5 liter of media. CHO cells weretransfected with either mouse tyrosinase or mutant variants, using thepcDNA3.1 expression vector and Lipofectamine LTX reagent according tothe manufacturer's instructions (Invitrogen, CA). In the cyclohexamide(CHX) experiments, cells were treated with CHX (2 μg/ml) or pretreatedwith tyrosine (1 mM) for 24 hours before addition of CHX in thetime-course assay (0, 1, 3, 6, 9, and 24 hours). Following the treatmentperiod, cells were harvested with lysis buffer (10 mM sodium phosphate,pH 7.0, 1% Igepal—CA630, protease inhibitor), and microfuged for 30minutes at 13,200×g, at a temperature of about 4° C., to obtain aprotein lysate. The total protein content in protein lysates wasdetermined spectrophotometrically as a 280/260 ratio. Protein expressionwas analyzed by Western blotting, and GAPDH was used as an internalloading control. For the analysis, Western blots were scanned,intensities of protein bands were determined, and ratios of bandintensities for the wild type or mutant variants, to that of GAPDH werecalculated. Care was taken in choosing non-saturated images foranalysis.

Melan-c cell cultures. Melan-c cells (melanocytes derived from micehomozygous for the albino mutation (D. C. Bennett et al., Development,105:379 (1989)) were cultured in RPMI 1640, pH 6.9 supplemented with 5%fetal bovine serum (FBS), streptomycin-penicillin (100 μg/ml each), 200nM tetradeconyl phorbol acetate (TPA), and 100 μM β-mercaptoethanol, at37° C. in 10% CO₂. Cells were transfected using Fugene HD reagentaccording to the manufacturer's instructions (Roche Diagnostics Inc.,Indianapolis, Ind.) in presence or absence of 1 mM tyrosine. After 24hours, cells were washed twice with saline phosphate buffer, harvestedin 10 mM sodium phosphate, pH 6.8, containing 1% Igepal CA-630 andprotease inhibitor (Roche), and microcentrifuged for 30 minutes at13,200×g, at a temperature of about 4° C. to obtain a protein lysate.

Diphenol oxidase activity of tyrosinase was determinedspectrophotometrically according to Slominski et al., J. Invest.Dermatol., 96:172 (1991), with minor modifications. Briefly, thereaction mixtures contained 7 mM L-dopa in 0.1 M sodium phosphate buffer(pH 6.8) and protein lysate (20 mg/ml) was incubated at 37° C. andmonitored by measuring the absorbance at 475 nm. All experiments wereconducted in triplicate.

Human melanocyte culture and melanin assay. Human melanocytes wereestablished from skin punch biopsies. Skin specimens were washed withPBS then treated with 0.25% trypsin-EDTA (Gibco 25200) for about 2 hoursfollowed by vigorous vortexing to separate the epidermis. The epidermiswas sectioned and attached to scored patches on the bottom of a 6-wellpolystyrene culture dish before being covered with melanocyte media.About 1000 ml of melanocyte media was made from 950 ml Ham's F10 (Gibco1550), 25 ml FBS, 5 ug bFGF (Sigma F0291), 10 μg endothelin (Sigma,E7764, Sigma Chemicals, St. Louis, Mo.), 7.5 mg IBMX (Sigma 17018), 30μg cholera toxin (Sigma C8052), 3.3 μg TPA (Sigma P8139), 10 mlPenStrepGlutamine, 1 ml fungizone and the media was 0.22 μm filtered.

Melanocytes were plated on 6 well dishes and grown to confluency.Melanin assays were run in triplicate by supplementing three wells perplate with 1 mM tyrosine (Sigma T8566), using the remaining 3 wells asuntreated controls. Treatment time was approximately one week.Melanocytes from each well were harvested separately by trypsinizationand washed twice with 1×PBS. Pellets were resuspended in 400 μl of 1×PBSand sonicated briefly. The lysate was then split to an equal volume of 2N NaOH (300 μl) and incubated at 80° C. for 1 hour to solubilizemelanin. The OD₄₇₅ was measured and converted to melanin content via astandard curve using synthetic melanin (Sigma M0418). The data werenormalized to protein content, determined using a bicinchoninic (BCA)assay kit (BioRad, Inc., Hercules, Calif.). Differences between treatedand untreated measurements were analyzed with a two-tailed unpairedt-test.

Study Participants. Research subjects with OCA were ascertained via anIRB-approved clinical research protocol at the National Human GenomeInstitute, National Institutes of Health. Human research was incompliance with the Declaration of Helsinki. OCA-1A and OCA-1B weredefined on clinical grounds based on hair, eye, and skin coloration atthe time of first clinical exam. In addition to decreased pigmentationin the hair and skin, both patients had ophthalmic abnormalitiesconsistent with albinism, including iris transillumination, nystagmus,decreased visual acuity, and an albinotic fundus, with no clear fovealreflex. Molecular confirmation included sequencing of the genes for OCAtypes 1 and 2 (TYR and OCA2 respectively). The OCA-1A subject had twoknown disease-causing mutations in TYR (c.230A>G, c.242C>T) and nolikely disease-causing variants in OCA2. The OCA1B subject had one knowndisease-causing variant (c.229T>A) in TYR, and no likely disease-causingmutation in OCA2 (up to 63% of OCA2B patients have no secondidentifiable TYR mutation).

Example 1

This example demonstrates tests the effect of clinically-relevant dosesof NTBC in a mouse model of oculocutaneous albinism, type 1a,(C57BL/6J-Tyr^(c-2J/c-2J)) using predefined ocular, systemic andbiochemical outcome variables.

C57BL6/J-Tyr^(c-2J/c-2J) mice are phenotypically albino due to a G291T(Arg77Leu) mutation in the Tyr gene that is functional null at theprotein level (Green, E. L., Mouse News Lett., 49:31 (1973)). These micehave a white coat color and pink eyes, and lack any significant funduspigmentation. As such, they are a reasonable model for oculocutaneousalbinism type OCA1a. Although these mice are completely albino, themutation in their tyrosinase gene is a missense mutation. This leavesopen the possibility that elevated tyrosine may stabilize the enzyme andimprove flux through pigment production pathways. To minimize the effectof additional genetic factors on phenotype, both lines of mice used inthese experiments are on the same inbred C57BL6/J background.

At the beginning of the study, baseline plasma concentrations oftyrosine, coat color (gross and microscopic), anterior segment pigment,and posterior segment pigment were documented for each mouse prior toinitiating experiments. At least ten C57BL6/J-Tyr^(c-2J/c-2J) mice, age3-4 months, were treated with about 25 μg NTBC in a volume of about0.2-0.3 ml, every other day, via oral gavage for a four-week period.These treated mice were compared to an age- and gender-matched cohort ofC57BL6/J-Tyr^(c-2J/c-2J) mice over the same time period. The efficacyand safety of NTBC at this dose was previously demonstrated in othermurine models. It is known that a possible side effect of NTBC treatmentis corneal irritation, which is monitored daily. At the start oftreatment, an area of hair was plucked or shaved from each mouse's back,which has the effect of stimulating new hair growth and possibly newpigment deposition in the hair shaft. At the end of four weeks time,plasma tyrosine concentrations were assessed in both treated and controlanimals.

Assessment of the effect treatment and the end of the experimental timeperiod commenced with photodocumentation of coat color, anterior segmentpigment and posterior segment pigment of each mouse. The mice weresacrificed using standard CO₂ euthanasia protocol. One eye from eachmouse was submitted for light microscopy, while the other was preparedfor electron microscopy. Liver and kidney tissues were collected andprocessed for light microscopy histology, to insure no pathology isassociated with treatment. Additional blood samples were taken andfrozen at the time of euthanasia, for possible future studies. Hairshafts in plucked and non-plucked areas were examined and photographedunder light microscopy. Melanosome number and size was quantified in theretinal pigmented epithelial cells (RPE) and choroid of the posteriorpole of both treated and untreated mice, in a masked fashion, aspreviously described by our group (Brooks, B. P., et al., Invest.Ophthalmol. Vis. Sci., 48(9):3905-13 (2007)).

Results. The initial animal protocol started with a dose of 1 mg/kg ofNTBC given to Tyr^(c-2J/c-2J) mice, every other day, by oral gavage, adose similar to that given to humans with tyrosinemia, type 1. TenTyr^(c-2J/c-2J) were treated and plasma tyrosine levels were ascertainedafter one month. At this dose, over this interval, no phenotypic changeswere noted and plasma tyrosine concentrations were not statisticallysignificantly different between treated and control animals.

Toxicologic studies show that male Alpk:ApfCD-1 albino mice (age 3-6weeks tolerate NTBC doses up to 160 mg/kg/day (Lock, E. A., et al.,Toxicology, 144:179-187 (2000)). Maximum plasma tyrosine concentrationswere achieved at or below 10 mg/kg NTBC. The dosing of NTBC in the twomouse models was increased to 4 mg/kg orally, every other day. Plasmatyrosine measurements of control and treated animals are presented inTable 1, below. NTBC treatment resulted in approximately a 6-foldincrease in steady-state plasma tyrosine concentrations inTyr^(c-2J/c-2J) mice.

The coat color was compared to representative control, and treated mice,at the end of the trial. Although plasma tyrosine levels were elevatedapproximately 6-fold, there was no difference in coat color, funduspigmentation, or iris transillumination in Tyr^(c-2J/c-2J) mice. Therewas also no observable change in the ocular pigmentation of control miceversus treated mice, over the time period studied (data not shown).

TABLE 1 Plasma Tyrosine Concentrations in Control and NTBC treated (4mg/kg q.o.d) Tyr^(c-2J/c-2J) and Tyr ^(c-h/c-h) Mice After 30 Days ofTreatment Group Tyr^(c-2J/c-2J) (OCA1a model) Tyr^(c-h) (OCA1b model)Control 109 ± 30 μM (n = 6) 74 ± 25 μM (n = 6) NTBC 673 ± 73 μM (n = 4,305 ± 35 μM (n = 4, Treatment p = 1 × 10⁻⁷) p = 2 × 10⁻⁶)

Example 2

This example demonstrates the effect of clinically-relevant doses ofNTBC in a mouse model of oculocutaneous albinism, type OCA1b,(C57BL/6J-Tyr^(c-h/c-h), carrying a temperature-sensitive mutation intyrosinase) using predefined ocular, systemic and biochemical outcomevariables.

The Himalayan mouse line is a mutant C57BL/6 mouse line which carries atemperature sensitive allele of tyrosinase, Tyr^(c-h) (MGI AccessionID#72456), that spontaneously arose in a C57BL/6 mouse in 1958, and hassince been inbred into the C57BL/6 background. The maximum activity ofthe tyrosinase produced from this allele occurs at temperatures belownormal body temperature (37° C.), because the mutant protein (c.A1259G,p.H420R) is heat labile. In homozygotes, the first coat is a uniformlight tan. At the first molt, the body hair becomes lighter and theears, nose, tail, and scrotum become dark as in Siamese cats. The eyesare slightly pigmented and appear red. Himalayan mice were housed instandard conditions, at room temperature. Because the Himalayan alleleretains some residual enzymatic activity, it is thought that these miceare a suitable model for type OCA1b oculocutaneous albinism.

Using the same protocol as described above, baseline plasma tyrosine,coat color (gross and microscopic), anterior segment pigment, andposterior segment pigment was documented for each mouse prior toinitiating experiments. At least ten C57BL/6J-Tyr^(c-h/c-h) mice, age3-4 months, were treated with about 25 μg NTBC in a volume of about0.2-0.3 ml, every other day, via oral gavage for a four-week period.These treated mice were compared to an age- and gender-matched cohort ofC57BL/6J-Tyr^(c-h/c-h) mice over the same time period. Cornealirritation due to NTBC treatment was monitored daily. At the start oftreatment, an area of hair was plucked/shaved from each mouse's back,which has the effect of stimulating new hair growth and possibly newpigment deposition in the hair shaft. At the end of four weeks' time,plasma tyrosine concentrations were assessed in both treated and controlanimals.

Assessment of the effect treatment and the end of the experimental timeperiod commences with photodocumentation of coat color, anterior segmentpigment and posterior segment pigment of each mouse. The mice weresacrificed using standard CO₂ euthanasia protocol. One eye from eachmouse was submitted for light microscopy, while the other is preparedfor electron microscopy. Liver and kidney tissues were collected andprocessed for light microscopy histology, to insure no pathology isassociated with treatment. Additional blood samples were taken andfrozen at the time of euthanasia, for possible future studies. Hairshafts in plucked and non-plucked areas were examined and photographedunder light microscopy. Melanosome number and size was quantified in theretinal pigmented epithelial cells (RPE) and choroid of the posteriorpole of both treated and untreated mice, in a masked fashion, aspreviously described above.

Results. As in Example 1 above, eight Tyr^(c-h/c-h) mice were treatedand plasma tyrosine levels were ascertained after one month. At thisdose, over this interval, no phenotypic changes were noted and plasmatyrosine concentrations were not statistically significantly differentbetween treated and control animals.

The dosing of NTBC in eight Tyr^(c-h/c-h) mice was increased to 4 mg/kgorally, every other day. Plasma tyrosine measurements of control andtreated animals are presented in Table 1. NTBC treatment resulted inapproximately a 4-fold increase in steady-state plasma tyrosineconcentrations in Tyr^(c-h/c-h) mice (data not shown).

It was found that biochemical changes were accompanied by phenotypicchanges in the Tyr^(c-h/c-h), but not the Tyr^(c-2J/c-2J) mice. Newpigment was deposited in hair shafts of the mice as they grew. Tostimulate this growth, a patch of hair on the back of control andtreated animals was shaved immediately prior to randomization.Photographs of the anterior and posterior segment ocular pigmentationwere also taken before and after the study period. While the coat andocular pigmentation of Tyr^(c-2J/c-2J) mice were grossly unchanged overthe one month period, there was a observable increase in pigmentation inthe treated Tyr^(c-h/c-h) mice compared to control Tyr^(c-h/c-h) mice.In some cases, pigmentation extended beyond the immediate area that wasshaved and other areas that were previously somewhat pigmented (e.g.,the nose) (data not shown).

While the irides of the Himalayan mice in the control group andpre-treatment showed a near complete absence of pigmentation, allanimals in the treated group showed some increase in iris pigmentation(data not shown). Fundus pigmenation was also grossly unchanged.However, when examined at the level of electron microscopy, preliminarydata showed an significant increase in the RPE and choroidal melanosomepigment content in treated, but not control mice (FIG. 3).

Example 3

In this example, a pediatric patient study for treatment of visionproblems associated with oculocutaneous albinism type OCA1b is provided.Patient recruitment begins with genotype testing to determine which typeof albinism each clinical subject. Patients are screened based on thegene that is mutated, for example, tyrosinase (OCA1), the P protein gene(OCA2), the tyrosinase-related protein-1 (TYRP-1, OCA3), and the MATPgene (OCA4). OCA1—the most common form of OCA in North AmericanCaucasians, can be further divided into those individuals who lacktyrosinase activity (OCA1a) and those who have some residual tyrosinaseactivity (OCA1b). Clinical molecular testing of the tyrosinase gene isused to identify mutations in patients who meet the clinical criteriafor albinism, but who make some pigment. The effect of the mutation onenzymatic activity is determined experimentally.

Vision assessment of all patients is undertaken to measure visualacuity, contrast sensitivity (with and without glare), reading speed,pigment production (via photography), nystagmus, strabismus andphotophobia, using standard methods in the art.

The patients are then randomized and assigned to blinded control andtreatment groups. The treatment groups are further divided into twodosage levels: 0.7 mg/kg/day and 1.0 mg/kg/day. The length of the studyis ninety days. Each week during the study, and at the end of the thirtyday trial, visual assessments of the patients are taken. In addition,observations of any other clinical manifestations of increasedpigmentation are noted. At the end of the study the visual measurementsof the patients in the two treatment arms are compared with those of thecontrol arm and significant statistical differences between the groupsare discerned.

Example 4

This example provides evidence that treatment with NTBC increasesmelanin content in the melanosomes of ocular tissues.

In order to quantitate the effect of NTBC on pigmentation in oculartissues and to assess for sub-clinical changes in ocular pigmentation,transmission electron microscopy (TEM) of iris, retinal pigmentepithelium (RPE), and choroid of treated and control mice (n=4 eyes from2 mice for each group) was performed. When TEM images of iris, choroidand RPE of NTBC-treated Tyr^(c-2J/c-2J) mice were compared with those ofuntreated mice, little to no increase in the number of pigmentedmelanosomes (stages III and IV) was observed, consistent with ourclinical observations (data not shown). The small amount of pigmentpresent in treated mice was irregular and not clearly in melanosomes. Incontrast, TEM images of iris, choroid, and RPE of NTBC-treatedTyr^(c-h/c-h) mice showed a clear increase in the number of pigmentedmelanosomes when compared with controls. This difference wasstatistically significant in all three tissues examined (FIG. 4).

Example 5

The following example describes how prenatal treatment with NTBCincreases coat and iris pigmentation in Tyr^(c-h/c-h) pups.

In order to assess whether elevation of plasma tyrosine by NTBCtreatment could have an effect early in development, pregnantTyr^(c-h/c-h) females were treated with 4 mg/kg NTBC. While pups ofvehicle-treated mothers had coat color similar to wild-type, the pups ofNTBC-treated mothers were considerably darker. Ocular examinationsperformed near the time of weaning showed that irides of pups born tovehicle-treated mothers resembled that of untreated Himalayan mice. Theirides of pups born from NTBC-treated mothers, however, showedsignificant pigmentation on clinical examination. There was nosignificant difference between the fundus appearance of pups born tovehicle-treated and drug-treated dams (data not shown). The pups oftreated mothers had no obvious congenital malformations or systemicillnesses. These data suggest that NTBC's effectiveness in increasingocular and cutaneous pigmentation in Himalayan mice extends into theprenatal/neonatal period.

Example 6

In the following example, in silico modeling of mouse tyrosinasemutations agrees with in vivo observations.

It was thought that NTBC exerted its pigment-increasing effect inHimalayan mice by increasing tyrosine concentrations, which, in turn,acts as a molecular chaperone for tyrosinase. In order to explain thediffering effects of NTBC on the two OCA models studied, the predictedeffect of the c-2J (R77L) and c-h (H420R) tyrosinase mutants weremodeled in silico.

The minimization procedure and molecular dynamics (MD) simulations wereperformed with the Impact module of the Maestro program package (version8.0.308, Schrodinger, Inc., New York, N.Y., USA). Hydrogen atoms wereadded to the structure of mouse tyrosinase and the structure wasregularized by an energy minimization procedure using the OPLS 2005potentials, the 12 Å non-bonded cut-offs, the distance-dependentdielectric constant and 100 steepest descent steps of minimizationfollowed by 200 steps of conjugated gradient in the presence of 7135 SPCwater molecules on the final step. MD trajectories were calculated in aperiodic rectangular box of explicit SPC water molecules. The structuresof the enzyme and EGF-like domains were equilibrated using the SPC waterbox with dimensions 70 Å×70 Å×70 Å for the tyrosinase enzyme domain andEGF-like motif. All bonds were constrained by the linear constraintsolver algorithm. The temperature was kept constant to 298.15 K.Isotropic pressure coupling of the system and fast particle-mesh Ewaldelectrostatics were applied. Solvent was equilibrated by 20 ps of solutepositions restrained MD (20 000 of 1 fs steps). Finally, the quality ofthe predicted structure was tested with the program Procheck (R. A.Laskowski, et al., J. Appl. Cryst., 26:283 (1993)).

Because X-ray crystallography has not been successfully performed onmammalian tyrosinase, the homology-modeling analyses previously-reportedand presented here rely, in part, on the available crystal structures ofprokaryotic (Streptomyces Castaneoglobisporus) and mushroom tyrosinase,invertebrate hemocyanin, and plant catechol oxidase (see, J. C.Garcia-Borron, et al., Pigment Cell Res., 15:162 (2002); W. P. Gaykema,et al., J. Mol. Biol., 187:255 (1986); T. Klabunde, et al., Nat. Struct.Biol., 5:1084 (1998); T. Schweikardt et al., Pigment Cell Res., 20:394(October, 2007); M. Sendovski, et al., J. Mol. Biol., 405:227 (2011); Y.Matoba, et al., J. Biol. Chem., 281:8981 (2006)).

The c-2J mutation, R77L, occurs in a structural fragment at the aminoterminus that is identified by the Simple Modular Architecture ResearchTool (SMART) (http://smart.embl-heidelberg.de/) as an EGF/laminin-likedomain. While the precise function of this domain is not known, the R77Lmutation is predicted to have major structural consequences based on thenegative blosum70 score (−3) and significant Grantham distance of 102.Structure equilibration using 3 ps molecular dynamics suggests that themutational change has a dramatic effect on tyrosine binding, which isthought to occur at the hydrophobic surface of the catalytic site. Assuch, it was thought that elevating ambient tyrosine concentrationswould have little to no effect on baseline enzyme function, in agreementwith in vivo results found.

In contrast, the Himalayan (c-h) mutation, H420R, demonstrates ablosum70 score of 0 and a smaller Grantham distance of 29, both of whichsuggest a less severe structural change. Rather than directly affectingthe structure of the hydrophobic tyrosine binding pocket, the modeldescribed herein predicts a greater effect on the coordination of coppernear the active site. As such, these results are consistent with in vivodata described herein.

Example 7

This example describes how elevated tyrosine concentrations stabilizeH420R, but not R77L, tyrosinase.

Given the in silico analysis described above, it was hypothesized thatH420R but not R77L tyrosinase can effectively bind tyrosine, and thatelevated ambient tyrosine could act as a molecular chaperone andselectively stabilize the Himalayan protein. In order to test thishypothesis, either wild-type, R77L, or H420R mutant tyrosinase proteinswere expressed in Chinese-hamster ovary (CHO) cells, and tyrosinaseprotein stability was measured using cycloheximide to inhibit newprotein synthesis. Similar levels of wild-type and mutant proteinexpression were observed on Western blots of cell protein lysates atbaseline (FIG. 5). As predicted, 1 mM tyrosine improved the stability ofthe H420R mutant protein (FIG. 6) at later time points (9 and 24 hours)relative to a marker protein, GAPDH. Although there was a trend towardsstabilization of the R77L mutant with 1 mM tyrosine, this was notstatistically significant (FIG. 7). These results agree with the in vivoobservations that pharmacological elevation of plasma tyrosine increasespigmentation in the Himalayan model of OCA-1B, but not in theTyr^(c-2J/c-2J) model of OCA-1A.

Example 8

In this example, elevated tyrosine concentrations result in pigmentproduction in melanocytes expressing OCA-1B, but not OCA-1A, Tyr allelesin vitro.

The ability of R77L and H420R mutant proteins to produce pigment invitro, compared to wild-type protein, in albino mouse melanocytes(Melan-c cells, D. C. Bennett et al., Development 105:379 (1989)) wasalso investigated. Mirroring our in vivo results, 1 mM tyrosineincreased enzyme activity over baseline in Melan-c cells expressing theH420R mutant tyrosinase (p=0.03), but not R77L mutant tyrosinase (FIG.8A).

In addition, the response of human melanocytes cultured from the skin ofOCA-1A and OCA-1B patients was studied. Similar to our results withtransfected mouse Melan-c cells, wild-type melanocytes producesignificant melanin in the presence of 1 mM tyrosine (FIGS. 8B, 8C).Melanocytes from an OCA-1A patient did not produce detectable pigmenteither in the presence or absence of 1 mM tyrosine. Melanocytes from apatient with OCA-1B showed baseline amounts of pigment similar tountreated controls. However, treatment with 1 mM tyrosine significantlyincreased pigmentation (p=0.0025), albeit not to the level observed inwild-type melanocytes. These in vitro results suggest that elevation ofcirculating tyrosine can increase pigmentation in humans with residualtyrosinase activity, implying that the effect observed in Himalayan micemay generalizable to other hypomorphic alleles of TYR/Tyr, and supportsthe idea that administration of an effective amount of NTBC to humanscan provide at least a temporary increase in the pigmentation of theskin for cosmetic uses.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-8. (canceled)
 9. A method of increasing of visually discerniblepigmentation in the eyes, hair or skin of a subject, the methodcomprising administering to the subject a pharmaceutical compositioncomprising (a) 2-(−2-nitro-4trifluoromethylbenzoyl)-1,3 cyclohexanedione(NTBC) or a pharmaceutically acceptable salt, hydrate, or solvatethereof, and (b) a pharmaceutically and physiologically acceptablecarrier in an amount effective to increase visually discerniblepigmentation in the eyes, hair or skin of the subject.
 10. The method ofclaim 9, comprising increasing plasma concentrations of tyrosine fromabout 7 micromolar (μM) to about 2 millimolar (mM) in the subject. 11.The method of claim 9, comprising increasing plasma concentrations oftyrosine from about 50 μM to about 300 μM in the subject.
 12. The methodof claim 9, comprising increasing plasma concentrations of tyrosine ofabout 70 μM in the subject.
 13. The method of claim 9, comprisingadministering NTBC to the subject in an amount between about 0.1mg/kg/day to about 10 mg/kg/day.
 14. The method of claim 9, comprisingadministering NTBC to the subject in an amount between about 0.5mg/kg/day to about 4 mg/kg/day.
 15. The method of claim 9, comprisingadministering NTBC to the subject in an amount between about 1 mg/kg/dayto about 2 mg/kg/day.
 16. The method of claim 9, comprisingadministering NTBC to the subject in an amount of about 1 mg/kg/day. 17.The method of claim 9, comprising increasing visually discerniblepigmentation in the eyes of the subject.
 18. The method of claim 9,comprising increasing visually discernible pigmentation in the hair ofthe subject.
 19. The method of claim 9, comprising increasing visuallydiscernible pigmentation in the skin of the subject.
 20. The method ofclaim 9, comprising increasing visually discernible pigmentation in theeyes, hair or skin for cosmetic purposes.